In the heart and its constituent muscle cells stretch is a major determinant of both the strength of cardiac contraction and the rate of secretion of protein hormones. We have shown that caveolae, abundant small vesicular invaginations of the plasma membrane, change their shape during stretch and what may be an analogous process, hyperosmotic shrinkage. These organelles may thus be part of the apparatus that transduces stretch signals in cardiomyocytes. During the previous grant period we have made significant progress on identification and purification of cardiac caveolae and their constituent proteins. Recently, we have made a breakthrough in this area by using an immunopurification protocol with antibodies directed against the muscle- specific caveolin-3 isoform. This improvement now allows us to ask a series of specific questions about the structure and function of cardiac caveolae and their relationship to perturbations such as stretch and osmolarity. There are three specific aims: Specific Aim 1 will (a) use immunoelectron microscopy to track the internalization of aquaporin1 from caveolae to an as yet unidentified locus in the atrial cytoplasm or elsewhere, (b) identify and localize the vesicles and microtubules involved, (c) look for and study the associated phosphorylations and signaling mechanism, and (d) determine if steps (a - c) manifest stretch dependence. Specific Aim 2 will (a) use a newly made rabbit polyclonal antibody to the muscle specific caveolin isoform, cav3 to optimize caveolar isolation from sheep and rat hearts; (b) use immuno-electron microscopy to identify immuno-purified caveolae, and standard analytic methods to identify specific lipid and protein components; (c) use microscale (e.g., electrospray) methods to define the protein composition of caveolae (d) study the signaling events in immunopurifed caveolae, and look for reversible phosphorylation, ADP-ribosylation, and effects of stretch. Specific Aim 3 examines the interaction of caveolae with the recently discovered extracellular matrix protein T-cadherin and the medically interesting caveolae-associated cytoskeletal protein dystrophin and its membrane anchor dystroglycan.